The present invention relates to oilseeds and oilseed plants capable of producing edible oils suitable for human consumption. More particularly, the invention relates to such seeds and plants capable of producing oils having desirable characteristics similar to those of canola oil, to processes of forming such plants, and to the resulting edible oils.
Brassica napus and Brassica rapa (campestris) are known as the canola species most effective for the production of edible oils for human consumption. These species set the benchmark in North America for Brassica oilseeds destined for the edible oil market. A Brassica oil of desirable quality must be of canola quality and contain no more than 2% by weight of the total fatty acids as erucic acid and no more than 30 xcexcmoles of aliphatic glucosinolates in the oil-free meal. The aliphatic glucosinolates of the meal can be any one or a mixture of 3-butenyl, 4-pentenyl, 2-hydroxy-3-butenyl or 2-hydroxy-4-pentenyl glucosinolate.
Canola was developed as an edible oil crop during the 1970xe2x80x2s by genetic alteration of rapeseed. Traditional rapeseed oil contains 25-45% by weight erucic acid and rapeseed meal contains 100-150 xcexcmoles of glucosinolates per gram, and is still grown and utilized as an edible oil and animal feed ingredient in a number of countries, primarily China and India. Lowering the erucic acid/glucosinolate levels to create canola led to the widespread use of the oil as a salad and frying oil and in the manufacture of margarine, shortening and other food products. In addition, the meal byproduct derived during the processing of canola seed is used as a high-protein feed ingredient in rations of poultry, swine, cattle and fish.
Canola oil has a fatty acid composition that is considered to be superior to many other vegetable oils for human nutrition (McDonald, 1995 [10]xe2x80x94please see the References section at the end of this description for full details of referenced articles). Canola is low in saturated fat, which has been shown to increase blood cholesterol levels and lead to increased risk of heart disease. Canola oil is high (55-60% by weight) in the mono-unsaturated fatty acid, oleic acid (C-18:1). Oleic acid has been shown to reduce serum cholesterol levels and is therefore desirable in an edible oil. An oil high in mono-unsaturated fat is more stable than an oil that is high in poly-unsaturated fatty acids, such as linoleic (C-18:2) and linolenic (C-18:3) (Eskin et al., 1989 [5]). Poly-unsaturated fatty acids are more easily oxidized during cooking, which creates off-flavours in the oil. Oxidation also reduces the shelf life of the oil.
The growing conditions of the Canadian Prairies are particularly suited to the cultivation of Brassica juncea, with approximately 50,000 hectares (ha) gown annually in this region of western Canada (Woods et al. [17]). However, this species of Brassica is grown to supply condiment mustard worldwide, and does not naturally produce an oilseed having a fatty acid content suitable for the production of an edible oil product. Specifically, oilseed of Brassica juncea naturally contain approximately 25% by weight of erucic acid and 100 xcexcmoles glucosinolates per gram of whole seed.
The main aliphatic glucosinolate in B. juncea grown as condiment mustard is 3-propenyl (allyl) glucosinolate, which is known to give mustard seed its hot and bitter taste. Further, the breakdown of this glucosinolate results in the formation of allyl isothiocyanate, which is believed to have detrimental effects on health (Ames, 1983 [2]).
Despite these characteristics, cultivars of B. juncea have been suggested as a potential source of edible oils on the basis of their improved resiliency and productivity over existing canola species. Specifically, cultivars of B. juncea are generally known to be high yielding, tolerant to both heat and drought, and disease resistant. Most particularly, B. juncea has shown superior resistance to important canola diseases such as blackleg. Some cultivars of B. juncea have also displayed resistance to pod shattering (Woods et al. 1991 [17]). Accordingly, the development of a canola-quality cultivar of B. juncea would help to increase and stabilize canola production, especially in hot, drought-prone regions.
Low erucic acid B. juncea germplasm was first identified in Australia (Kirk and Oram, 1981 [7]). These lines were designated as Zem 1 and Zem 2 and were released to plant breeders. Agriculture and Agri-Food Canada (AAFC) initiated a plant breeding program in 1985 to develop B. juncea canola, using the Zem lines as a starting point.
Researchers at AAFC developed a low glucosinolate line using an interspecific cross with a low-glucosinolate B. rapa line (Love et al., 1990 [8]). A line designated 1058 was developed that had less than 10 xcexcmoles of total glucosinolates per gram of meal, but had very low fertility, low oil content and high erucic acid content. This initial line was improved upon and lines were developed with improved yield, higher oil content and low erucic acid content (Love et al., 1991 [9]; Rakow et al., 1995 [12]).
Low erucic acid B. juncea lines developed in Australia and by AAFC were more unsaturated (containing more linoleic and linolenic acid) than canola cultivars of B. napus or B. rapa. Oil of these lines is considered to be of lower quality than normal canola, and therefore difficult to integrate into the mainstream canola crop (Raney et al., 1995 [13]).
Researchers have attempted to alter the fatty acid profile of B. juncea through interspecific crosses. Raney et al. (1995) [13] crossed low erucic B. juncea with low linolenic B. napus. A single backcross to B. juncea was made and plants were selfed (self-fertilized). In F4 generation seed, the highest oleic value was 53.7% by weight, and the saturated fat level was 9.7% by weight (palmitic and stearic). This fatty acid profile would be unacceptable as canola. Nevertheless, all generations from the backcross had depressed fertility and poor seed set, indicating that there was genetic instability as a result of the interspecific cross.
Agnihotri et al. (1995) [1] derived lines from a cross of (Eruca sativaxc3x97B. rapa)xc3x97B. juncea. One line was reported to have an oleic content of 61.9% by weight; however, this line had 103.4 xcexcmoles of allyl glucosinolate per gram of meal, so it would not be considered to be canola quality. As described, the genetic makeup of this line was only 50% B. juncea and the generation was not specified, so genetic stability was not proven. It is highly unlikely that an interspecific line such as this would have good fertility and all of the agronomic characteristics associated with B. juncea. 
International Patent Application PCT/US96/02620 to Pioneer Hi-Bred International, Inc., (published on Sep. 12, 1996 under International Publication No. WO 96/27285) discusses the potential use of a B. napus line to produce B. juncea that is low in linolenic acid, high in oleic acid and low in saturated fatty acids. Evidence of a B. juncea plant with an altered fatty acid profile produced by this or any other means is totally absent. No claim in the application refers to B. juncea. 
Despite all these efforts, the need remains for a B. juncea line which displays superior agronomic qualities and produces an endogenous oil with an acceptable level of oleic acid (at least 55% by weight), which is low in erucic acid (less than 2% by weight), low in saturated fat (the total of C-16:0, C-18:0, C-20:0 and C-22:0 less than 7.1% by weight), low in total aliphatic glucosinolates (less than 30 xcexcmoles per gram of meal) and low in allyl glucosinolate (less than 3 xcexcmoles per gram of meal). To be useful, the line must be genetically stable, must have acceptable agronomic performance compared to current canola species and must have retained the positive attributes of B. juncea, such as adaptation to a semi-arid environment and resistance to blackleg.
An object of the present invention is to provide a line of Brassica juncea, and the resulting oilseeds, that satisfies the above requirements to acceptable extents.
Another object of the invention is to provide a process of producing such lines of Brassica juncea. 
Another object of the present invention to provide a novel mature B. juncea oilseed capable of yielding an edible endogenous vegetable oil having a distribution of fatty acids that would be accepted as a canola equivalent oil following simple crushing and extraction.
Yet another object of the present invention to provide a novel B. juncea plant having satisfactory agronomic characteristics which upon self-pollination is capable of yielding an endogenous vegetable oil having a distribution of fatty acids that would be acceptable as a canola equivalent oil following simple crushing and extraction.
It is another object of the present invention to provide an edible vegetable oil extracted from B. juncea seeds that meets the major specifications for canola oil, as listed in Food Chemicals Codex (1996) and as required by the Canadian canola industry.
It is a further object of the present invention to provide a process for the formation of genetically stable, agronomically adapted B. juncea cultivars, wherein the distribution of the major fatty acids meets the specifications for canola oil as listed in Food Chemicals Codex (1996).
According to one aspect of the invention, there is provided an oilseed of the species Brassica juncea bearing an endogenous oil having an oleic acid content of at least 55% by weight, a linoleic acid content of less than 25% by weight, a linolenic acid content of less than 14% by weight, a erucic acid content of less than 1% by weight, a palmitic acid content of less than 6% by weight, a stearic acid content of less than 2.5% by weight, and a total saturated fatty acid content of less than 7.1% by weight.
According to another aspect of the invention, there is provided a genetically stable plant of the species B. juncea that develops mature seeds bearing an endogenous oil having an oleic acid content of at least 55% by weight, a linoleic acid content of less than 25% by weight, a linolenic acid content of less than 14% by weight, a erucic acid content of less than 1% by weight, a palmitic acid content of less than 6% by weight, a stearic acid content of less than 2.5% by weight, and a total saturated acid content of less than 7.1% by weight; or a part or precursor of said plant.
According to another aspect of the invention, there is provided a seed oil having an oleic acid content of at least 55% by weight, a linoleic acid content of less than 25% by weight, a linolenic acid content of less than 14% by weight, a erucic acid content of less than 1% by weight, a palmitic acid content of less than 6% by weight, a stearic acid content of less than 2.5% by weight, and a total saturated acid content of less than 7.1% by weight, said oil having been extracted from an oilseed of a line of the species Brassica juncea. 
According to yet another aspect of the invention, there is provided a process of producing a genetically stable B. juncea plant that develops mature seeds bearing an endogenous oil having an oleic acid content of at least 55% by weight, a linoleic acid content of less than 25% by weight, a linolenic acid content of less than 14% by weight, a erucic acid content of less than 1% by weight, a palmitic acid content of less than 6% by weight, a stearic acid content of less than 2.5% by weight, and a total saturated acid content of less than 7.1% by weight, said process comprising the steps of: crossing a line of B. juncea having a lineage that includes J90-3450 with a B. juncea line having a lineage that includes J90-4316 to form F1 progeny; propagating said progeny by self-pollination or by producing doubled haploid progeny and, from resulting progeny, selecting genetically stable plants that generate seeds containing an endogenous oil that has an oleic acid value of more than 55% by weight, a linoleic value of less than 25% by weight, a linolenic acid value of less than 14% by weight, an erucic acid value of less than 1% by weight, a palmitic acid value of less than 6% by weight, a stearic acid content of less than 2.5% by weight and a total saturate content of less than 7.1% by weight.
According to yet another aspect of the invention, there is provided a process of producing a genetically stable B. juncea plant that develops mature seeds bearing an endogenous oil having an oleic acid content of at least 55% by weight, a linoleic acid content of less than 25% by weight, a linolenic acid content of less than 14% by weight, a erucic acid content of less than 1% by weight, a palmitic acid content of less than 6% by weight, a stearic acid content of less than 2.5% by weight, and a total saturated acid content of less than 6.5% by weight, said process comprising the steps of: crossing a line of B. juncea having a lineage that includes J90-3450 with a B. juncea line having a lineage that includes J90-4316 to form F1 progeny; propagating said progeny by a method selected from the group consisting of either self-pollination or development of doubled haploid plants; and, from resulting progeny, selecting genetically stable plants that generate seeds containing an endogenous oil that has an oleic acid value of more than 55% by weight, a linoleic value of less than 25% by weight, a linolenic acid value of less than 14% by weight, an erucic acid value of less than 1% by weight, a palmitic acid value of less than 6% by weight, a stearic acid content of less than 2.5% by weight and a total saturate content of less than 7.1% by weight; and utilizing mutagenesis to produce a plant with a low saturated fat content, crossing the low saturated plant or its progeny with a plant with  greater than 55% by weight oleic acid to produce progeny with an oleic acid value of more than 55% by weight, a linoleic value of less than 25% by weight, a linolenic acid value of less than 14% by weight, an erucic acid value of less than 1% by weight, a palmitic acid value of less than 6% by weight, a stearic acid content of less than 2.5% by weight and a total saturate content of less than 6.5% by weight.
Thus, by means of the present invention, an endogenous vegetable oil extracted from Brassica juncea is provided which exhibits an oleic acid content of at least 55% by weight, a linoleic acid content of less than 40% by weight, a linolenic acid content of less than 14% by weight, a erucic acid content of less than 2% by weight, a palmitic acid content of less than 6% by weight, a stearic acid content of less than 2.5% by weight, and a total saturate content of less than 7.1% by weight. The oil is essentially equivalent in fatty acid composition to canola oil extracted from B. napus or B. rapa 
In a preferred embodiment, B. juncea cultivars of the present invention also possess an aliphatic glucosinolate content of less than 30 xcexcmoles per gram of oil-free meal. The allyl glucosinolate content is preferably less than 3 xcexcmoles per gram of oil-free meal.
Brassica juncea plants are provided that are genetically stable, are morphologically recognizable as B. juncea, have generally the same desirable agronomic attributes as mustard-quality B. juncea and produce an endogenous oil that is essentially equivalent in fatty acid composition to canola oil.
A process is provided for the formation of B. juncea plants that produce endogenous oil that has a total saturate content of less than 6.5% by weight. This process includes the use of a mutagenic agent to produce DH plants with lower saturated fat content and then crossing said plants with high oleic B. juncea plants to produce a high oleic B. juncea plant with reduced saturated fat content.